The X-linked lymphoproliferative disease gene product SAP associates with PAK-interacting exchange factor and participates in T cell activation

Abstract

SLAM (signaling lymphocyte activation molecule)-associated protein (SAP) is a Src homology 2 (SH2) domain-containing adaptor expressed in T cells and natural killer cells. Its essential role inimmune responses is underscored by the recent finding that mutations in SAP result in a rare but fatal X-linked lymphoproliferative disease (XLP). Although SAP is known to associate with SLAM-family receptors, the exact molecular mechanism by which SAP regulates lymphocyte signaling remains elusive. We here report that in T cells, SAP associates with the PAK-interacting exchange factor (PIX), a guanine nucleotide exchange factor (GEF) specific for Rac͞Cdc42 GTPases. Moreover, SAP, PIX, and an activated form of Cdc42 form a complex in mammalian cells. We demonstrate that the SAP-PIX interaction is specific and is mediated by the C-terminal region of the SAP SH2 domain and the PIX SH3 domain. We further show that SAP is required for the recruitment of PIX to the SLAM-family receptors. Interestingly, overexpression of SAP, but not its homolog EAT-2, leads to a synergistic activation of nuclear factor of activating T cells (NFAT) in combination with a calcium signal in T cells. This SAP-mediated activation appears to be receptor-dependent and can be blocked by a dominant negative form of PIX. Taken together, our data strongly suggest that, in addition to the known SAP-interacting kinase Fyn, PIX may be another key player in SAP-mediated T cell activation.nuclear factor of activating T cells (NFAT) ͉ Cdc42 ͉ 2B4 ͉ Fyn X -linked lymphoproliferative disease (XLP) is an inherited severe or fatal immune dysfunction generally triggered by infection with Epstein-Barr virus (EBV) (1). XLP patients often develop a lethal fulminant infectious mononucleosis after EBV infection. Some patients also develop progressive dysgammaglobulinemia, malignant lymphomas, or autoimmunity such as vasculitis (2-4). Several immune cell types have been shown to exhibit functional alterations in XLP, including CD4 ϩ T cells, CD8 ϩ T cells, natural killer cells, and B cells (3). In 1998, three groups identified the gene aberrant in XLP, named as SAP͞ SH2D1A͞DSHP (5-7). The human SLAM (signaling lymphocyte activation molecule)-associated protein (SAP) is comprised of 128 amino acids, of which residues 6-102 contribute to an N-terminal SH2 domain. Many experiments have shown that the SH2 domain of SAP preferentially recognize specific tyrosinebased motifs containing TxYxxV͞I that are present within the cytoplasmic tails of SLAM and its related receptors, including 2B4, CD84, Ly9, NTB-A, and CRACC (1,8,9). Because XLP patients lack functional SAP, it has been speculated that the phenotypes of XLP likely reflect perturbed signaling downstream of one or more of these receptors, ultimately resulting in abnormal immune responses.Given the severe clinical manifestations in XLP patients, recent efforts have been focused on elucidating the signaling mechanism by which SAP regulates in T and natural killer cells. It has been shown that SAP recruits a Src kinase Fyn to...

Expanding functions of GIT Arf GTPase-activating proteins, PIX Rho guanine nucleotide exchange factors and GIT–PIX complexes

“…In Tcells, activation of the T-cell receptor by antigen leads to activation of PAK1 through a GIT-PIX complex, which together are recruited to the immune synapse (Ku et al, 2001;Phee et al, 2005). The SAP adaptor protein, a T-cell signaling protein that is mutated in Xlinked lymphoproliferative disease, binds to the SH3 domain of PIX to couple SLAM receptors to Cdc42 activation during T-cell activation (Gu et al, 2006). Accordingly, mice deficient in α-PIX or GIT2 exhibit immune abnormalities.…”

“…Similarly, Rac1 and/or Cdc42, and their PAK effectors have been reported to bind to or affect GIT-PIX. But, in addition to PIX binding to inactive Rac1 and/or Cdc42 during activation, active Rac1 and/or Cdc42 (and other Rho family members) have been reported to also bind to GIT-PIX (Brown et al, 2002;Shin et al, 2004;Gu et al, 2006;ten Klooster et al, 2006;Hajdo-Milasinovic et al, 2009;Tay et al, 2010;Wilson et al, 2014). The effects binding of active Rho family proteins has to GIT-PIX remain illdefined and appear somewhat contradictory because GIT and PIX have each been reported to directly bind Rac1 and/or Cdc42 in cellbased studies where both are present, clouding interpretation.…”

A Novel Interaction between Atrophin-interacting Protein 4 and β-p21-activated Kinase-interactive Exchange Factor Is Mediated by an SH3 Domain

“…The ␤PIX-SH3 domain also binds to the PXXXPR motif of Cbl family members to facilitate the clustering of proteins involved in the down-regulation of EGF receptor signaling (26,28). Interestingly, the ␤PIX-SH3 domain also binds proteins, including Rac1 and SAP (signaling lymphocyte activation molecule-associated protein) (29,30) that possess neither the PXXXPR nor the canonical PXXP motifs. The structural basis for these interactions and the functional ramifications of competition among multiple ligands for overlapping binding sites on ␤PIX remain to be elucidated.…”

SLAM Family Receptors and Autoimmunity

“…Furthermore, SAP can additionally engage Lck, which phosphorylates CD84, CD150, CD229 and CD244 (Howie et al, 2002;Martin et al, 2005;Nakajima et al, 2000;Tangye et al, 2003). The SAP-SH2 domain has also been described as interacting with the SH3 domain of PAK-interacting protein ( -PIX), a guanine nucleotide exchange factor (GEF) specific for Rac/Cdc42 GTPases (Gu et al, 2006). Both human and murine EAT-2 genes are located at chromosome 1 (1q23 in humans and 1H3 in mice), in close proximity to SLAMF loci (Calpe et al, 2006).…”